Application Note: Microbial Genomics
Metagenomic Analysis of Environmental
Water Samples With the NextSeq 500 System
Shotgun metagenomic sequencing provides insight into microbial responses to environmental
changes in a water reservoir.
Introduction
Environmental metagenomics is the study of organisms in a microbial
community by analyzing the DNA present in an environmental
sample. The advent of next-generation sequencing (NGS) technology
has enabled researchers to profile entire microbial communities in
complex samples quickly and easily. Unlike capillary electrophoresis
or PCR, NGS allows investigators to sequence thousands of
organisms in parallel. With shotgun metagenomic sequencing,
microbiologists can examine the genes of the organisms in a given
sample comprehensively to evaluate bacterial diversity and detect
microbial abundance in various environments. When designing a
shotgun metagenomic sequencing study, researchers must consider
many factors, including the complexity of the sample and the
sequencing output required to assess microbial diversity. Illumina
library preparation kits and the NextSeq Series of sequencing systems
delivers the efficiency, high throughput, speed, and sample size
flexibility needed for efficient and affordable metagenomics studies.
This application note describes how investigators from the Center
for Earth and Environmental Science and Indiana University-Purdue
University Indianapolis (IUPUI) used shotgun metagenomic sequencing
to characterize microbial communities in samples from the Eagle
Creek reservoir in Indianapolis, Indiana. The method includes sample
collection, DNA extraction using the Meta-G-NomeTM DNA Isolation
Kit,1 DNA library preparation with the Nextera® XT DNA Library Prep
Kit,2 sequencing with the NextSeq 500 System,3 and analysis using
the MG-RAST metagenomics analysis server4 (Figure 1). The results
presented in this study provide new insights into the taxonomic and
functional differences between microbial communities in the Eagle
Creek drinking water supply reservoir at different times and locations.
Sample Extraction Library Preparation
Figure 1: Shotgun Metagenomic Sequencing Workflow—The speed of the Nextera XT DNA Library Prep Kit and the output of the NextSeq Series contribute to a
fast and efficient workflow suited to NGS-based environmental metagenomics studies.
For Research Use Only. Not for use in diagnostic procedures.
®
Methods
Sample Collection
In collaboration with Citizens Water Company,5 the Center for Earth
and Environmental Science (IUPUI) regularly samples, monitors, and
documents the water supply in the Eagle Creek reservoir. The samples
for this study were collected at regular intervals, from May to October
2013, before the first frost. Discrete samples were collected from the
surface, and at the depths of 3 meters, 6 meters, and near the bottom
floor of the reservoir. During the sampling process, 150 ml of lake
water were collected, filtered through a 0.22 μm filter, and frozen
at -20°C.
Genomic DNA Isolation
After sample collection, DNA was isolated directly from the water
sample using the Meta-G-Nome DNA Isolation Kit (Epicentre®,
an Illumina company). The protocol uses filtration technology and
enzymatic lysis to isolate DNA from the water sample. The kit
is designed to isolate randomly sheared genomic DNA of high
molecular weight.
Library Preparation
The Nextera XT DNA Library Prep Kit was used to construct libraries
from the isolated DNA. Using a single enzymatic “tagmentation”
reaction, the Nextera transposome simultaneously fragmented and
tagged the DNA with unique adapter sequences. Limited-cycle PCR
was used to amplify the tagged DNA and add sequencing indexes.
Using this streamlined workflow, 11 DNA libraries were prepared for
sequencing on the NextSeq 500 desktop sequencer in half a day.
Sequencing Analysis
Application Note: Microbial Genomics

Sequencing Table 1: Sequencing Data

All 11 libraries were pooled together for cluster generation and Metagenome Sequence
MG-RAST ID Base Counta
sequencing. Libraries were loaded onto a reagent cartridge and Name Countb
clustered on the NextSeq 500 System. A paired-end, 2 × 150 bp 4554374.3 1305-530 5,061,868,863 33,395,202
sequencing run was performed using the NextSeq 500 High-Output 4554375.3 1305-531 5,566,909,825 37,789,429
Kit. A single sequencing run generated 400 million reads in 29 hours, 4554376.3 1305-532 8,409,674,885 57,196,837
corresponding to an average of 40 million reads per sample after
4554377.3 1305-533 4,341,474,376 31,003,711
quality filtering (Table 1). Base calls generated by the NextSeq 500
4554378.3 1307-521 5,184,242,473 32,673,383
System were converted to FASTQ files for metagenomic analysis.
4554379.3 1307-522 14,173,422,835 85,796,130
Data Analysis 4554380.3 1307-523 8,369,469,023 52,993,984
4554381.3 1307-524 2,864,009,160 19,306,387
Data analysis is often the most challenging part of a metagenomic
sequencing study. Several programs and pipelines are available to 4554382.3 1310-522 8,688,763,557 55,503,302
perform metagenomic analysis. These programs are optimized for 4554383.3 1310-523 10,943,344,357 69,814,786
different study objectives, such as taxonomic profiling, assessing 4554384.3 1310-524 7,776,089,825 46,838,779
microbial composition, or identifying functional genes and pathways.
a. All sequenced bases
b. Number of sequencing reads
The analysis presented in this study used the publicly available
MG-RAST program, an automated analysis platform that provides
various tools for data visualization enabling users to assess species
composition or functional abundance. FASTQ files generated by the
NextSeq 500 System and sample metadata (Table 2) were uploaded
to the MG-RAST server. Overlapping paired-end reads were joined
and then processed in MG-RAST using the default trimming and
filtering settings to allow comparison to other data sets in MG-RAST.

Table 2: Environmental Metadata

Water
Location Salinity
MG-RAST ID Sampling Depth Sampling Date Temperature pH Air Temperature (oC)
Coordinatesa (ppm)
(oC)
4554374.3 Surface 23 May 2013 21.05 8.72 13.3 39.827, -86.303 0.21
4554375.3 3 meters 23 May 2013 21.00 8.73 13.3 39.827, -86.303 0.21
4554376.3 6 meters 23 May 2013 13.77 8.27 13.3 39.827, -86.303 0.22
4554377.3 Floor 23 May 2013 10.78 7.50 13.3 39.827, -86.303 0.20
4554378.3 Surface 25 July 2013 26.50 8.48 18.9 39.826, -86.304 0.21
4554379.3 3 meters 25 July 2013 26.02 8.40 18.9 39.826, -86.304 0.23
4554380.3 6 meters 25 July 2013 23.03 7.60 18.9 39.826, -86.304 0.22
4554381.3 Floor 25 July 2013 13.86 7.16 18.9 39.826, -86.304 0.23
4554382.3 3 meters 23 October 2013 15.09 7.80 4.4 39.826, -86.304 0.24
4554383.3 6 meters 23 October 2013 15.06 7.72 4.4 39.826, -86.304 0.24
4554384.3 Floor 23 October 2013 14.99 7.68 4.4 39.826, -86.304 0.24

a. Global Positioning System (GPS) coordinates

For Research Use Only. Not for use in diagnostic procedures.

 Application Note: Microbial Genomics

Arthrobacter
Rhodococcus
Synechococcus
Renibacterium
Clavibacter
Bacillus
Leifsonia
Mycobacterium
Streptomyces
Actinobacteria)
Cyanobium
Sanguibacter
Methylobacter
Kocuria
Lysinibacillus
May Jul May Jul Oct May Jul Oct May Jul Oct

Figure 2: Comparative Genus Abundance Over Time—Comparisons of relative genus abundance (shown for the 15 most abundant genera) demonstrated a sharp
decline in Arthrobacter populations on the reservoir floor in July 2013. These data were generated using MG-RAST.

Results Learn More

Analysis of species abundance with MG-RAST revealed drastic To learn more about the NextSeq Series, visit
changes in microbial composition on the reservoir floor and an www.illumina.com/nextseq.
increase in the Rhodococcus genus (a soil bacteria), at the 3 meter
For more information about the use of Illumina technology in microbial
depth in July 2013 (Figure 2). For the reservoir floor, the population
genomics, visit www.illumina.com/microbiology.
declines might be correlated with an algaecide treatment that occurred
on 31 May 2013. It is possible that the copper in the algaecide
treatment caused the sharp decline in species from the Arthrobacter References
genus. This hypothesis is based on the observation that Arthrobacter 1. Meta-G-Nome- DNA Isolation Kit (www.epibio.com/applications/nucleic-
globiformis often forms a symbiotic relationship with the Anabaena acid-purification-extraction-kits/dna-purification-genomic/meta-g-nome-
genus of nitrogen-fixing cyanobacteria and the aquatic ferns in the dna-isolation-kit) Accessed 21 April 2014.
Azolla genus, both part of the algae family.6 After algaecide treatment, 2. Nextera XT DNA Library Prep Kit (www.illumina.com/products/nextera_xt_
both Arthrobacter globiformis and Azolla experienced a decline; dna_sample_prep_kit.ilmn) Accessed 21 April 2014.
3. NextSeq 500 Desktop Sequencer (www.illumina.com/systems/nextseq-
however, Azolla is not shown in Figure 2, because it is not 1 of the 15
sequencer.html) Accessed 21 May 2015.
most abundant genera in the samples. The hypothesis that the
4. Meyer F, Paarmann D, D’Souza M, et al. The metagenomics RAST server -
gram-positive Anabaena species might have played a role in the
a public resource for the automatic phylogenetic and functional analysis of
changes to community composition requires further analysis. Likewise metagenomes. BMC Bioinformatics. 2008;9:386.
for the 3 meter sampling depth, where analysis revealed an increase 5. Citizens Energy Group (www.citizensenergygroup.com) Accessed 21
in the Rhodococcus genus, further analysis is required to assess April 2014.
root cause. 6. Martens T, Gram L, Grossart HP, et al. Bacteria of the Roseobacter
clade show potential for secondary metabolite production. Microb Ecol.
2007;54:31-42.
Conclusions
This study demonstrates how shotgun metagenomic sequencing
with the NextSeq 500 System can reveal information about the
microbial composition of a particular environment. The data presented
show a correlation between the environmental metadata and the
species composition in the Eagle Creek reservoir. This study provides
new insights into the biological processes potentially associated
with algal blooms, sampling depth, and seasonal differences in
freshwater environments.

For Research Use Only. Not for use in diagnostic procedures.

Application Note: Microbial Genomics

Illumina • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com

For Research Use Only. Not for use in diagnostic procedures.
© 2014–2015 Illumina, Inc. All rights reserved. Illumina, Epicentre, Meta-G-Nome, Nextera, NextSeq, and the pumpkin orange color are
trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. Pub. No. 1270-2014-002 Current as of 26 May 2015